CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

Abstract

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Assigning functionality to cysteines by base editing of cancer dependency genes

“…The copyright holder for this preprint this version posted November 17, 2022. ; https://doi.org/10.1101/2022.11.17.516964 doi: bioRxiv preprint proteins. Recently, an alternative HDR-based oligo recombineering approach was described to assess the functionality of cysteines in Toxoplasma gondii 63 . This approach is well-suited for focused, array-based screens, particularly in haploid systems, but currently lacks the throughput and efficiency to perform large-scale analyses of thousands of cysteines in diploid mammalian cells.…”

“…We were also fortunate that the ABE and CBE systems produced non-conservative mutations of cysteines (Cys-to-Arg or Cys-to-Tyr) that were anticipated to have good potential to alter the functions of proteins. Recently, an alternative HDR-based oligo recombineering approach was described to assess the functionality of cysteines in Toxoplasma gondii 63 . This approach is well-suited for focused, array-based screens, particularly in haploid systems, but currently lacks the throughput and efficiency to perform large-scale analyses of thousands of cysteines in diploid mammalian cells.…”

A solid-phase compatible silane-based cleavable linker enables custom isobaric quantitative chemoproteomics

“…Chemoproteomic studies, including our own 15,17,47,48,[53][54][55] , continue to rely on stable isotope incorporation, either through metabolic labeling or the aforementioned custom isotopically labeled capture reagents, and MS1 based quantification 10,42,[56][57][58] . These methods are widely adopted in large part due to their relatively reasonable cost, the compatibility of the resulting datasets with freely available software packages for analysis [59][60][61] , and increased data reproducibility afforded by the ability to combine 'treated' and 'control' samples early in the sample preparation workflow.…”

Bioinspired 5-caffeoylquinic acid capped silver nanoparticles using Coffee arabica leaf extract for high-sensitive cysteine detection

“…Interaction studies between polyphenols and amino acids are having a great concern in day-to-day life because it reveals the idea behind protein-phenol complex formation and mechanisms [42][43] . For example, Lund et al reported the higher a nity of thiol group in cysteine for CGA o-quinones which was a complements knowledge of polyphenol-protein interaction that applied in sun ower protein food products which is recognized as one of top 6 plant-based food products 44 .…”